Immunotherapy for reversing immune suppression

ABSTRACT

A method for overcoming immune suppression includes the steps of inducing production of naïve T cells and restoring T cell immunity. A method of vaccine immunotherapy includes the steps of inducing production of naïve T cells and exposing the naïve T cells to endogenous or exogenous antigens at an appropriate site. Additionally, a method for unblocking immunization at a regional lymph node includes the steps of promoting differentiation and maturation of immature dendritic cells at a regional lymph node and allowing presentation of processed peptides by resulting mature dendritic cells, thus, for example, exposing tumor peptides to T cells to gain immunization of the T cells. Further, a method of treating cancer and other persistent lesions includes the steps of administering an effective amount of a natural cytokine mixture as an adjuvant to endogenous or exogenous administered antigen to the cancer or other persistent lesions; preferably the natural cytokine mixture is administered in combination with thymosin α 1 .

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. Section119(e) of U.S. Provisional Patent Application No. 60/344,509, filed Oct.26, 2001, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to vaccine therapy for cancer patients andpatients having persistent lesions, such as infections. Morespecifically, the present invention relates to a vaccine immunotherapythat immunizes patients, having immune suppression, to both endogenousand exogenous tumor peptides or proteins, as well as those derived fromother persistent lesions.

2. Background Art

It has become increasingly apparent that human cancers have antigens,which, if reacted upon by the host's immune systems, lead to tumorregression. These antigens have been defined by both serological andcellular immune approaches. This has led to the definition of both B andT cell epitopes (Sahin, U, et al., Curr Opin Immunol, 9:709-715 (1997);Van der Eynde, B, et al., Curr Opin Immunol, 9:684-693 (1997); Wang, RF, et al., Immunologic Reviews, 170:85-100 (1999)). Based upon theseresults, it has become a goal of cancer immunotherapists to induceregressions of tumors. However, historically, successful efforts havebeen sporadic and generally minor in frequency and magnitude.

A fundamental problem in the effort to immunize cancer patients is thatthe tumor-bearing state is associated with immunosuppressive mechanismsderived from both the tumor and the host's disturbed immune system(Kavanaugh, D Y, et al., Hematol-Oncol Clinics of North Amer.,10(4):927-951 (1996)), thereby making immunization difficult, and untilnow, impossible on a consistent basis. Immune suppression or depletioninvolves a reduced capacity of the immune system to respond. Suchsuppression can be drug or disease induced. The condition can be druginduced by treatment, virus induced as in AIDS, or induced by a diseasestate such as cancer. The immune system in this condition is effectivelyturned off.

A variety of tumor immunization strategies have been developed. However,all of these strategies are complex and deviate significantly from theconventional immunization strategies used for infectious diseases(Weber, J Tumor Medscape Anthology, 3:2 (2000)). One such tumorimmunization strategy involves Theratope®, a Sialyl T_(N) polysaccharidemucin antigen conjugated with keyhole limpet hemocyanine andadministered with Detox® mycobacterium adjuvant and low dosecyclophosphamide (Maclean G D, et al., J Immunother Emphasis TumorImmunol., 19(4):309-316 (1996)). However, use of this vaccine inpatients with metastatic breast and ovarian cancers has yielded majorclinical responses in a low percentage of patients. A major responsemeans greater than 50% tumor reduction.

Gene therapy also has been attempted using an adenovirus construct as anexpression vector for genes expressing Papilloma virus. Peptide 16 hasbeen used for immunization for patients with cervical cancer and hasyielded major clinical responses in a low percentage of patients(Borysiewickz, L K, et al., Lancet, 347:1524-1527 (1996)).

Dendritic cell mediated therapy also has been attempted, whereindendritic cells were pulsed with oligopeptide fragments of prostatespecific antigens (PSA). Prostate specific membrane antigen (PSMA) hasbeen used in patients with metastatic prostate cancer with majorclinical responses in a low percentage of patients (Sanda, M G, et al.,Urology, 52:2 (1999); Murphy, G P, et al., The Prostate, 38:43-78(1999)).

Additionally, autologous tumors have been used with low dosecyclophosphamide and BCG to immunize cancer patients with malignantmelanoma. However, few clinical responses were reported (Mastrangelo MJ, et al., Seminars in Oncology, 23(6):773-781 (1996)). Another strategyattempted included using MAGE antigens with a variety of vaccineadjuvants. Again, this has yielded few, if any, responses in patientswith malignant melanoma.

Several U.S. patents to Doyle, et al., (U.S. Pat. Nos. 5,503,841;5,800,810; 6,060,068; 5,643,565; 5,100,664) disclose methods ofenhancing the immune response in patients using Interleukin-2 (IL-2).This method is disclosed for use in response to infectious diseases andprimarily functions using antigens known to be immunogenic. Limitedapplicability was demonstrated. As disclosed above, the treatment ofcancer is known to require different approaches. To date, treatment withIL-2 has shown minor effects in two cancers, renal cell and malignantmelanoma (response rates less than 20%). It is generally consideredineffective in squamous cell head and neck cancer, cervical cancer, andin prostate cancer. Hence, it is not approved for these uses. It wouldtherefore not be within the skill of one in the art to apply the methodof the Doyle, et al. patents to the use of small peptides in thetreatment of cancer.

It is important to contrast prevention with known “classic” antigens ofcomplex structure and high molecular weights in healthy patients versustreatment (generally unsuccessful) with tumor antigens or peptides(generally unsuccessful) in immunosuppressed patients (generallyunsuccessful). The first is easy and current viral vaccines attest totheir efficacy. The latter is nearly impossible on a routine basisdespite 30 years of intense effort.

It is important that this invention relates to, but not exclusively to,immunizing with endogenous peptide processed and presented by dendriticcells or endogenously administered to an environment (lymph node) wheredendritic cells have been prepared and can present them to T cellseffectively. This goal is considered by many immunologists to beinsurmountable. Peptides are much too small to be effective immunogens,their half-life is short, they are often nonmutated self-antigens towhich the patient is immunologically tolerant, and gaining a response istantamount to inducing autoimmunity.

In several of the above strategies, cellular and/or tumoral immunity totumor-associated antigens has been induced (Weber, J Tumor MedscapeAnthology, 3:2 (2000); Maclean, G D, et al., J Immunother Emphasis TumorImmunol, 19(4):309-316 (1996); Borysiewickz, L K, et al., Lancet,347:1524-1527 (1996); Sanda, M G, et al., Urology, 52:2 (1999)). This isespecially so in association with tumor regression. Nevertheless, thesuccess rate of such treatments is negligible and inconsistent (<30%).

It would therefore be useful to develop a consistent and effectivemethod of immunizing cancer patients.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a method forovercoming immune depression by inducing production of naïve T cells andrestoring T cell immunity. That is, the present invention provides animmune restoration. The present invention further provides a method ofvaccine immunotherapy including the steps of inducing production ofnaïve T cells and exposing the naïve T cells to endogenous or exogenousantigens at an appropriate site. Additionally, the present inventionprovides a method for unblocking immunization at a regional lymph nodeby promoting differentiation and maturation of immature dendritic cellsat a regional lymph node and allowing presentation of processed peptidesby resulting mature dendritic cells, thus exposing tumor peptides to Tcells to gain immunization of the T cells. Additionally, the presentinvention provides a method of treating cancer and other persistentlesions by administering an effective amount of a natural cytokinemixture as an adjuvant to endogenous or exogenously administered antigento the cancer or other persistent lesions.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention are readily appreciated as thesame becomes better understood by reference to the following detaileddescriptions when considered in connection with the accompanyingdrawings, wherein:

FIG. 1 is a graph showing a comparison of NCM in different mediautilizing continuous versus pulsed exposure to PHA;

FIG. 2 is a graph showing the effect of cell concentration withcontinuous exposure to PHA;

FIG. 3 is a bar graph similar to FIG. 1 with PHA at twice theconcentration (2 micrograms per ml);

FIG. 4 is a graph of thymidine uptake versus units per ml of IL-2relating to splenocytes;

FIG. 5 is a graph similar to FIG. 2 related to thymocytes;

FIG. 6 is a graph showing ratio to control versus in vivo treatments formice with involuted thymuses treated with IL-1, IL-2 or IL combinations,NCM, or saline;

FIG. 7 is a graph also showing a comparison of treatment withrecombinant IL-1, IL-2, IL-1 plus IL-2, and NCM;

FIG. 8 is a graph demonstrating the effect of NCM treatment in vivo onsplenocyte and thymocyte markers;

FIG. 9 is a bar graph also demonstrating the effect of NCM treatment invivo on splenocyte and thymocyte markers;

FIG. 10 is a graph demonstrating splenocyte and splenocyte responses toin vitro media, including various recombinant interleukins or NCM aftertreatment in vivo with control media or NCM;

FIG. 11 is a bar graph demonstrating the splenocyte and thymocyteresponses in vitro to media, various interleukins, or NCM in vivo withcontrol media or NCM;

FIG. 12 demonstrates responses in splenocyte and thymocyte in vitro toConA and PHA after treatment in vivo with control or NCM;

FIG. 13 demonstrates responses in splenocyte and thymocyte in vitro toConA and PHA after treatment in vivo with control or NCM;

FIG. 14 is a bar graph showing node size in controls, and cancercontrols or IRX-2 (NCM) treated populations with squamous cell head andneck cancer (H&NSCC);

FIG. 15 shows two bar graphs, the first showing T cell area and thesecond showing density in controls and head and neck squamous cancercontrols and patients treated with NCM (IRX-2);

FIG. 16 shows two bar graphs showing B cell area and follicles in thethree treatment groups;

FIG. 17 shows a comparison of other cells and sinus histiocytosis in thethree treatment groups;

FIG. 18 is a graph showing node B and T and cancer B and T fit plot:

FIG. 19 shows increases of lymphocyte populations in the blood of IRX-3treated patients induced by a 10-day treatment of NCM plus thymosin α1;

FIG. 20 shows increases in naïve T cells (CD45RA+) and memory T cells inthe blood of IRX-3 treated patients induced by a 10-day treatment of NCMplus thymosin α1;

FIG. 21 is a bar graph of thymocyte response in vitro to media (openbar). rIL-1 (closed bar), rIL-2 (cross-hatched), and NCM (diagonallines) after treatment in vivo with saline, thymosin α1 (5μg/animal/day), NCM (50 units IL-2 equivalence) and thymosin α1 (5μg/animal/day)+NCM (50 units IL-2 equivalence); and

FIG. 22 is a bar graph of splenocyte responses in vitro to media (openbar), rIL-1 (closed bar), rIL-2 (cross-hatched) and NCM (diagonal lines)after treatment in vivo with saline, thymosin α1, NCM and thymosinα1+NCM as in FIG. 21.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention provides methods for treating patientsutilizing vaccine immunotherapy wherein the patients are immunesuppressed. By immune suppressed, it is meant that the patient hasreduced cellular immunity and thus impaired capacity to respond to newantigens.

T lymphocytopenia (low T cell levels in blood) is a diagnosticcharacteristic of cellular immune deficiency; impaired function ofexisting lymphocytes is the other characteristic. There is no generallyaccepted (clinically approved) way to treat T lymphocytopenia. Bonemarrow transplants (±thymus transplants) have been used in cases ofsevere combined immunodeficiency (SCID—congenital, irradiation orchemotherapy induced). Recombinant IL-2 has been tried in AIDS with someeffect by much toxicity.

There are two ways to make new T cells to attempt to correct Tlymphocytopenia. One way, as in rIL-2 therapy, expands T cells alreadyin the periphery, i.e., memory T cells (CD45RO) (blood, lymph node andspleen). The other involves processing in the thymus of new T cells frombone marrow-derived precursors. This happens naturally in children butnot in adults. These new cells are called recent “thymic émigrés” andhave the surface marker of “naïve” T cells, i.e., CD45RA. NCM therapy(plus thymosin α1) results in the production of these new T cells aswell as expanding pre-existing memory T cells.

More specifically, the present invention utilizes new discoveriesrelating to immunization to provide an immune response to antigens thatis either endogenously or exogenously administered. Such antigens in thepast have been believed to be immunogenic while others used in thepresent invention have been previously thought to be nonimmunogenic.Examples of such antigens are EADPTGHSY (melanoma) from MAGE-1 protein,EVDPIGHLY (lung carcinoma) from MAGE-3, EVDPIGHLY (lung carcinoma) fromMAGE-3, and many others (see, Bellone, et al., Immunology Today,20(10):457-462 (1999)).

The present invention utilizes several general newly derived methodsteps for obtaining immunization in subjects where such immunization waspreviously thought to be impossible. More specifically, the presentinvention provides a method for overcoming severe immune depression byinducing production of naïve T cells. The term “naïve” T cells is meantto mean newly produced T cells, even in adults, wherein these T cellshave not yet been exposed to antigen. Such T cells at this stage arenonspecific yet capable of becoming specific upon presentation by amature dendritic cell having antigen, such as tumor peptides, exposedthereon. Thus, the present invention replenishes or generates new Tcells. This is generally accomplished by administering a naturalcytokine mixture (NCM). The NCM includes IL-1, IL-2, IL-6, IL-8, IL-10,IL-12, δIFN, TNFα, and both G- and GM-CSF plus thymosin α1. The amountand proportions of these constituents are detailed below. Preferably,about 150-600 units of IL-2 are contained in the NCM and 1.6 mg ofthymosin α1.

Preferably, the NCM with thymosin α1 is injected around lymphatics thatdrain into lymph nodes regional to a lesion, such as tumor or otherpersistent lesions being treated. Perilymphatic administration into thelymphatics that drain into the lymph nodes, regional to the lesion, suchas a cancer, is critical. Peritumoral injection has been associated withlittle response, even progression, and is thus contraindicated. A 10-dayinjection scheme is optimal and a 20-day injection protocol, whileeffective clinically, tends to reduce the TH1 response and shift towardsa less desirable TH2 response as measured by lymphoid infiltration intothe cancer. Bilateral injections are effective. Where radical neckdissection has occurred, contralateral injection is effective.

It is preferable to block endogenous suppression of T cells, such ascaused by various cancer lesions. Blocking is effected by the codeliveryof low dose cyclophosphamide and a nonsteroidal anti-inflammatory drug(NSAID). The NSAID of choice is indomethacin. While indomethacin is themost effective NSAID, it is also arguably the most toxic. Celebrex® andVioxx®, Cox II NSAIDs, are less effective. Vioxx® can be more toxic,causing gastritis in many patients. Ibuprofen was effective but thehistological responses were characteristic of a TH2 rather than TH1mediated response, this being less desirable. Side effects of NSAIDs areto be aggressively treated with proton inhibitors and a prostaglandin Eanalog. Zinc and multivitamins are useful agents to help restore T cellimmunity. Treatment with contrasuppression and zinc without the NCM isineffective.

In summary, the minimum regimen is perilymphatic treatment with the NCMplus thymosin α1 combined with contrasuppression using cyclophosphamideand an NSAID. The alternative regimen is the previously mentionedregimen further including zinc and vitamins, possibly including theaddition of selenium. Preferable dosing is zinc 50 to 75 mg. A standardmultivitamin can be administered. The zinc can be an availablegluconate.

In order to maximize clinical response, and for the greatest increase insurvival rate, the degree and type of lymphocyte infiltration isimportant. Granulocyte or macrophage infiltration of a 90:10 ratio isoptimal. T and/or B cell infiltration preferably is diffuse and intenseand not peripheral. Light infiltration of less than 20% is notassociated with a robust clinical response. Tumor reduction andfragmentation in the histological samples are preferred in reflecting agood response.

Lymph node changes key to good response involve at least five aspects.Lymph node enlargement, and not just reversal of tumor induced reductionof size but overall increase in size compared to normal, is preferred.Increased T and B cell areas indicate an immunization. The present dataindicate that sinus histiocytosis (SH) is an accumulation ofnonactivated mature dendritic cells (CD68+CD83+DC) that have ingestedand processed tumor antigens but are unable to mature and present thesetumor peptides to naïve T cells capable of stimulating TH1 and TH2effective cells that lead to cytotoxin T and B cells. Reversal of SH andthe activation of DC is a key to the invention.

Thus, the present invention provides for unblocking immunization at aregional lymph node by promoting maturation and activation of dendriticcells in a regional lymph node and thus allowing presentation byresulting mature dendritic cells of small peptides, generally nine aminoacids in length, to T cells to gain immunization of the T cells, asdiscussed in greater detail below. Additionally, induction of maturedendritic cells is required. Finally, mobilization of peripheral blood Tlymphocytes in T lymphocytopenic patients in the presence of inductionof naïve T cells capable of responding to dendritic cells presentingendogenous tumor peptides is desired (see, Sprent, et al., Science,293:245-248 (2001)).

In view of the above, the key mechanistic features of the presentinvention are the in vivo maturation of dendritic cells resulting ineffective peptide antigen presentation. Based on the examples presentedherein, increases in CD45RA positive naïve uncommitted T cells have beenfound. This leads to T and B cell clonal expansion, creating immunity inthe patient. The resulting infiltration into tumors by hematogenousspread leads to robust tumor destruction. The result, as found in thedata herein, is increased survival due to immunologic memory (see,Sprent, et al.).

It is predicted logically that exogenously provided synthetic orextracted tumor peptides (see, Bellone, et al.) can be delivered intothe preprimed or coprimed regional or distal lymph node and yield tumorantigen specific T cells, with or without B cells. Examples are setforth below. In view of the above, it can be concluded that the actionof NCM and thymosin α1 plus other agents is useful as for any tumorantigens (synthetic and endogenous, peptides and proteins). Many ofthese peptides are not normally immunogenic and only when presented bymatured, activated dendritic cells, will they be effective in immunizingnaïve T cells. Thus, the appearance of an immune T cell means, de facto,that a dendritic cell has been made or allowed to work properly. Also defacto, dendritic cell activation and maturation are considered keyfactors in cancer immunodeficiency, as well as the well-known defects inT cells, such as a decreased number and function with anergy andpresumed apoptosis.

Referring more specifically to the protocol and medicant delivered inaccordance with the present invention, the invention utilizes thenatural cytokine mixture plus thymosin α1 to immunize patients, such ascancer patients, as well as patients with other lesions or antigenproducing disease conditions. More specifically, the present inventionutilizes a method of enhancing the immune response of cancer patients toa cancer by administering an effective amount of a compositioncontaining therein the NCM plus thymosin α1 and a tumor-associatedantigen, the NCM plus thymosin α1 acting as an adjuvant to produce theimmune response.

The tumor-associated antigen can be either an endogenously processedtumor peptide preparation resident in regional nodes of patients withcancer, or in conjunction with an exogenously administered tumor antigenpreparation in or near these nodes. Tumor peptides, as well as antigens,are included herein even though peptides are not expected to beimmunogenic where tumor-associated protein antigens would be more likelyso since they are complete.

In the preferred embodiment, the composition of the present inventioninvolves the administration of the NCM plus thymosin α1 plus atumor-associated or specific antigen, as defined below with low doses ofcyclophosphamide, a cyclooxygenase inhibitor, and other similarcompounds that have been shown to further increase the effects of thecomposition of the present invention. The NCM and thymosin α1combination is an adjuvant creating an immune response to antigens nototherwise found to be effectively antigenic. Moreover, this adjuvanteffect has been accomplished in patients who are severely immunedeficient.

To clarify and further define the above, the following definitions areprovided. By “adjuvant,” it is meant a composition with the ability toenhance the immune response to a particular antigen. To be effective, anadjuvant must be delivered at or near the site of antigen. Such abilityis manifested by a significant increase in immune mediated protection.Enhancement of immunity is typically manifested by a significantincrease (usually greater than 10-fold) in the titer of antibody raisedto the antigen. Enhancement of cellular immunity can be measured by apositive skin test, cytotoxic T cell assay, ELISPOT assay for δIFN orIL-2, or T cell infiltration into the tumor as described herein.

By “tumor associated antigen,” it is meant an analogous protein orpeptide (which were previously shown to work by pulsing of dendriticcells ex vivo) or other equivalent antigen. This can include, but is notlimited to, PSMA peptides, MAGE peptides (Sahin, U, et al., Curr OpinImmunol 9:709-715 (1997); Wang, R F, et al., Immunologic Reviews170:85-100 (1999)), Papilloma virus peptides (E6 and E7), MAGEfragments, or other similar antigens. Previously, these antigens werenot considered to be effective in treating patients based either ontheir size, i.e., they are too small or that they were previouslythought to not have the immunogenic properties (i.e., self-antigens).

NCM, a nonrecombinant cytokine mixture, is defined as set forth in U.S.Pat. Nos. 5,632,983 and 5,698,194. Briefly, NCM is prepared in thecontinuous presence of a 4-aminoquinolone antibiotic and with thecontinuous or pulsed presence of a mitogen, which in the preferredembodiment, is PHA.

Pooled lymphocytes, generally from the buffy coat, free of neutrophilsand erythrocytes from HIV-negative, hepatitis virus-negative multipledonors are used to produce a mixed lymphocyte response (MLR). Further,in a preferred embodiment, up to 50 donors are used each time to producethe mixture to ensure that the MLR response is constant for eachpreparation and to even out variation.

In an alternative embodiment, autologous lymphocytes are used togenerate the NCM. In these cases, the patient does have to bevirus-free. Further, if autologous lymphocytes are used, they can bereturned to the patient as needed. In an alternative embodiment, animalscan be the cell source for veterinary uses.

The lymphocytes are cultured in the presence of immobilized mitogens ina tissue culture vessel. In a preferred embodiment, the mitogen isimmobilized on surface activated cell culture flasks for selection ofcell subsets (AIS MICROCELLECTOR™ T-25 plates) as described in themanufacturer's instructions. However, other methods of immobilizingmitogens on the surface of the culture vessel such as methodsincorporating other “panning” techniques or coupling to sepharose 4Bbeads could be used as are well known in the art of cell isolation. Theuse of immobilizing cells for selection is well known in the art.

The mitogens are generally selected from lectins and monoclonalantibodies that stimulate lymphocytes to produce cytokines. In apreferred embodiment, phytohemagglutinin (PHA) or OKT3 (Orthoclone®,Ortho Pharmaceuticals) are used. Other lectins such as concanavalin A(ConA) or pokeweed mitogen that stimulate B cells can be used.Monoclonal antibodies to T cell receptors such as CD2, CD28, CD45 can beused as mitogens. Anti-CD28 and CD45 antibodies are reported to behyperproducers of IL-2 (Deans, et al., (1989); June, et al., (1989).Further, antilymphocyte globulin (ALG) has mitogenic activity for Tcells. In addition, combinations of mitogens can be used to activate acombination of lymphocyte subpopulations. PHA is used in the preferredembodiment and is coated at a starting concentration of about 25 μg/ml.

The lymphocytes are incubated for 24 to 28 hours in a serum-free mediawith continuous exposure to the mitogen, i.e., no washings. In apreferred embodiment, the media is either X vivo-10 or X vivo-15 media(Whittaker). This is a serum-free and FDA-approved media for IL-2/LAKinfusions in patients as set forth in the manufacturer's brochure.Serum-free media capable of supporting human lymphocyte proliferation,such as RPMI-1640 (Sigma), can be also used.

The media also contains a 4-aminoquinolone antibiotic. In the preferredembodiment, the antibiotic is Ciprofloxacin. The antibiotic is used tomaintain sterility and to hyperproduce lymphokines. Ciprofloxacin andrelated antibiotics have been reported to increase IL-2 and othercytokines in the presence of the soluble mitogen and serum (Riesenbeck,et al. (1994)). They have not been reported to be effective in theabsence of serum. Ciprofloxacin is used in the preferred embodiment at aconcentration of from about 20 to about 200 μg/ml and more preferably,at a concentration of about 80 μg/ml.

The supernatant is removed and is the source of the NCM of the presentinvention. The supernatant is free of the mitogen as shown in Example 1.In animal and initial human studies. it does not have to beconcentrated.

Human serum albumin (HSA) can be added to stabilize the NCM in thesupernatant. HSA is used instead of serum albumin from a nonhuman sourcebecause HSA has been approved by the FDA for human use.

A cytokine profile of the supernatant is established utilizing thefollowing assays. The interleukin content of the supernatant isconfirmed by bioassay for IL-2 and by ELISA's for other interleukins,CSFs, TNFs, and IFNs. Sterility is tested and endotoxin measured bylimulus lysate assay. Specifically, the following assays and kits areused in a preferred embodiment: INF-γ ELISA (ENDOGEN), IL-1, IL-2, IL-3,IL-4, IL-6, IL-7, IL-8, GM-CSF, G-CSF, and TNF-α ELISAs (R&D Systems).The IL-2 bioassay of Gillis, et al., 1978, is expressed as units/mlcompared to a known standard of IL-2 (Schiapparelli Biosystems, Inc.,Fairfield, N.J.).

In the preferred embodiment, wherein PHA is used as the mitogen, thecytokine profile for the supernatant has a profile of:

CYTOKINE AMOUNT IL-1 10-2000 pg/ml IL-2 100-500 units/ml IL-6 250-10,000pg/ml IL-8 12,000-100,000 pg/ml IL-12 100-10,000 pg/ml IFN-γ 50-15,000pg/ml TNF-α 50-15,000 pg/ml CSF-G 50-1,500 pg/ml CSF-GM 10-1,500 pg/mlIL-3/IL-4/IL-7 Trace Amounts

Immobilization of the mitogen produces a higher yield of NCM than doespulse techniques. For example, production of interleukins by a pulsetechnique with PHA in serum-free media yielded IL-2 at 0-20 units/mlmedia (U.S. Pat. Nos. 4,390,623 and 4,464,355). However, the presentmethod allows an increased production with a pulse technique by adding a4-aminoquinolone antibiotic to the serum-free media to hyperinduceinterleukin and yielded about 8-140 units/ml of IL-2. As predicted bythe animal studies, this preparation, characterized as a naturalinterleukin mixture (NIM), at 200 units IL-2/dose, increased Tlymphocyte counts in blood of lymphopenic patients with head and neckcancer (Hadden, et al. (1994)). This result has not been reported atdoses greater than 5,000 times the amount of IL-2 in NCM. Thus, it isimportant to note that the dose IL-2 equivalent for NCM is used as anindex of its potency and is not meant to imply that the total biologicalactivity of NCM is that of only IL-2.

In the preferred embodiment of the present invention, utilizingcontinuous exposure to the mitogen by immobilization and the presence ofa 4-aminoquinolone antibiotic, the NCM that is generated generallycontains IL-2 at 100-353 units/ml (an index of the potency of thepreparation). In the less preferred embodiment, the invention can bepracticed with the continuous presence of 4-aminoquinolone antibioticand a pulsed presence of the mitogen, producing NIM. This combinationproduces a level of cytokines greater than the other prior art methodswith a pulsed mitogen only, but does not produce the levels seen withthe preferred embodiment of the present invention.

The invention can be also practiced with a natural nonrecombinantinterleukin mixture (NIM) that is produced with the continuous presenceof 4-aminoquionlone antibiotic but with only a pulsed presence of amitogen such as PHA. Other immunomodulating natural nonrecombinantcytokine preparations such as an NIM preparation, also can be used inthe present invention. The various preparations are compared by IL-2content, and the dosage is referred to as IL-2 equivalents.

Thymic peptides are used in the present invention coadministered withthe immunomodulator-cytokine preparations. Thymosin α1 (T-α1), or itsanalogs and fragments, is used in the preferred embodiment of thepresent invention. In addition, other thymic peptides, such as thymosinα11 and prothymosin and their analogs can be used. Thymic peptides,analogs, and fragments that contain the thymosin α1 sequence can be alsoused.

An analog will be generally at least 70% homologous over any portionthat is functionally relevant. In more preferred embodiments, thehomology will be at least 80% and can approach 95% homology to thethymic peptide, particularly the thymosin al sequence. The amino acidsequence of an analog may differ from that of the thymic peptide when atleast one residue is deleted, inserted, or substituted. Differences inglycosylation can provide analogs. Analogs as set forth in U.S. Pat.Nos. 4,116,951; 4,353,821; 4,466,918; 4,470,926; 4,612,365; and4,910,296 are examples of such analogs and can be used in the presentinvention.

A partially characterized NCM has been previously shown to be effectivein promoting T cell development and function in aged, immunosuppressedmice. Thymosin α1 also protected T cell development and function in agedimmunosuppressed mice and the combination of NCM plus thymosin α1 wasdramatic in its action to produce new T cells in the spleen (U.S. Pat.No. 5,698,194). Upon administering this NCM to immunosuppressed patientswith head and neck cancer, it is demonstrated in this application forthe first time that the mobilization of T lymphocytes in the blood ofcancer patients treated with the NCM produces an increase in immature,naïve T cells bearing both CD2 and CD45RA. This is one of the firstdemonstrations that adult humans can generate naïve T cells. It isdescribed in this application that NCM plus thymosin α1 producesincreased “naïve” T cells in irradiated patients resistant to NCMtreatment. Previous references (Mackall, et al., New Eng J Medicine332:143-149 (1995); and a review by Mackall, Stem Cells 18:10-18 (2000))discuss the inability to generate new T cells in adults but notchildren, and discuss the problem of trying to replenish T cellsfollowing cancer chemotherapy and/or radiotherapy. In general, there isthe dogma that new T cells are not generated in the adult human.However, following bone marrow transplantation for intense chemotherapy,there has been evidence that new T cells can be generated in the adult.No molecular therapy to date has been able to achieve this, although anincrease in lymphocyte counts have been achieved with prolonged andintense therapy with recombinant interleukin-2 in patients infected byHIV. These have not been clearly demonstrated to be thymus-derived Tcells and are presumably an expansion of pre-existing peripheral Tcells.

Previously, Cortesina, et al., employed a natural IL-2,perilymphatically, in patients with head and neck cancer and inducedseveral tumor regressions (Cortesina G, et al., Cancer 62:2482-2485(1988)) with some tumor infiltration with leukocytes (Valente, G, etal., Modern Pathol 3(6):702-708 (1990)). Untreatable recurrencesoccurred and the response was termed nonspecific and without memory andthus nonimmunologic (Cortesina, G, et al., Br J Cancer 69:572-577(1994)). The repeated attempts to confirm the initial observations withrecombinant IL-2 were substantially unsuccessful (Hadden, J. W., JImmunopharmacol, 11/12:629-644 (1997)).

The method of the present invention involves using NCM plus thymosin α1with local perilymphatic injections or other injections that are knownto those of skill in the art to provide sufficient localization of theimmunotherapy compound. In the preferred embodiment, the injections takeplace in the neck, but can be applied in other locations as required bythe disease to be treated. This treatment induced clinical regressionsin a high percentage of patients who also showed improved,recurrence-free survival (Hadden, J W, et al., Arch Otolaryngol HeadNeck Surg 120:395-403 (1994); Meneses A, et al., Arch Pathol Lab Med122:447-454 (1998); Barrera, J, et al., Arch Otolaryngol Head Neck Surg,126:345-351 (2000); Whiteside, et al., Cancer Res 53:564-5662 (1993)),observed that in head and neck cancer, tumoral injection of recombinantinterleukin-2 produced a T cell lymphocyte infiltrate, but withoutsignificant clinical responses. Peritumoral injection of Multikine(Celsci website) in combination with perilymphatic injection in up to150 patients resulted in significant tumor responses, i.e., greater than50% tumor reduction in only 11 patients, making their response rate lessthan 10% in contrast to the high degree of response observed in thepresent studies, 40%. In addition, they noted 50% nonresponders whereApplicant has observed only 20%.

Applicant has observed that peritumoral and intratumoral injection canbe associated with progression of disease even in patients who initiallyhave had a positive response to the NCM protocol, thus undoing itsbenefit. Peritumoral injection is thus contraindicated and is excludedas part of the present invention. This has led Applicant to theinterpretation that the tumor is not the site of immunization and thepresent application presents documentation that the regional lymph nodeis the site of immunization. Then, unpublished analysis of regionallymph nodes revealed data that indicated that the regional lymph node isthe site of immunization to postulated tumor antigens (FIGS. 14-18).With the identification of a number of different tumor antigens, it hasbeen a conundrum over the last decade that given the presence of suchantigens, they have not been employed effectively in immunizationprotocols. Sporadic positive examples have been reported, but in themain, the data are negative. The problem of antigen presentation hasbeen focused on in the last decade and the dendritic cell has emerged asa critical player in the presentation of small peptides derived fromtumors (DeLaugh, et al., Curr Opin in Immunol 12:583-588 (2000);Buchereau, et al., Ann Rev of Immunol 18:767-811 (2000); Albert, et al.,Nature 392:86-89 (1998)).

In brief, in order for tumor antigens to be properly antigenic, theymust arrive from an apoptotic rather than a necrotic tumor cell (Albertreference in Nature). They need to be captured by immature dendriticcells that have the morphology of large histocytes. These immaturedendritic cells process (endocytosis, phagocytosis and digestion) andevolve into mature dendritic cells that display peptide fragments(generally nine amino acids) of the digested antigen in the MHC groovefor presentation to T cells. T cells, in order to respond, must haveantigen presented to them in the MHC groove plus various costimulatorysignals (Banchereau and DeLaugh).

Investigators, such as Murphy, et al. (1999), have utilized dendriticcells generated in culture and then pulsed with tumor antigens and haveachieved a small degree of success in immunizing patients againstprostate specific membrane antigen peptides. Unfortunately, thisapproach of pulsing dendritic cells is cumbersome and has been ratherinefficient. Herein, Applicant has shown that the cells present in thelymph node sinuses, which accumulate in cancer, are cells of the lineageof dendritic cells. Following the in vivo treatment with the NCMprotocol, these cells disappear and antigen ultimately then becomesimmunogenic for T cells. They are able then to respond to the tumor.Therefore, a critical aspect of this invention is being able to generatea microenvironment in the regional lymph node that allows effectiveantigen processing and presentation. The immunization derives T cellsable to traffic to the lesion and destroy tumors. This is de factodemonstration of adequate antigen processing by dendritic cells.Additionally, none of the patients treated with NCM developed distantmetastasis that is expected in up to 15% clinically and up to 50%pathologically. This indicates that a systemic immunity rather thanmerely a local immunity has been induced by the treatment. This is adrastic improvement over the compositions in the prior art, because theprior art compositions, at best, were inconsistently effective againstmetastatic disease. The ability of the composition of the presentinvention to create systemic immunity allows more effective andefficient treatment of a patient.

The literature (Hadden, J W, J Immunopharmacol 11/12:629-544 (1997);Hadden, J W, Immunology and Immunotherapy of Breast Cancer: An Update,Int'l J Immunopharmacol 21:79-101 (1999)) has indicated that for bothSCC and adenocarcinomas, the two major types of cancer, regional lymphnodes reflect abnormalities related to the tumor, including sinushistiocytosis, lymphoid depletion and often the presence of anergictumor-associated lymphocytes (capable of reacting to tumor cells with exvivo expansion and recovery using IL-2). Then, with metastases, lymphoiddepletion and depressed function occur. Additionally, uninvolvedcervical lymph nodes of such patients have shown a reduction in averagesize and an increase in sinus histiocytosis associated with head andneck cancers. (See FIGS. 14 through 17).

The composition of the present invention involves the natural cytokinemixture plus either endogenous or exogenous tumor-associated antigen.Additionally, low doses of cyclophosphamide, cyclooxygenase inhibitors,zinc, and other similar compounds have been shown to further increasethe effects of the composition of the present invention.

Immunization for treatment of patients with cellular immune deficienciesassociated with cancer, HIV infection, aging, renal transplants, andother such deficiencies can be achieved with the composition of thepresent invention.

Administration and protocols for treatment follow.

Delivery of Gene Products/Synthetic Antigens:

The compounds of the present invention (including NCM) and exogenousantigens are administered and dosed to achieve optimal immunization,taking into account the clinical condition of the individual patient,the site and method of administration, scheduling of administration,patient age, sex, and body weight. The pharmaceutically “effectiveamount” for purposes herein is thus determined by such considerations asare known in the art. The amount must be effective to achieveimmunization, including but not limited to, tumor reduction,fragmentation and infiltration, survival rate or more rapid recovery, orimprovement or elimination of symptoms.

In the method of the present invention, the compounds of the presentinvention can be administered in various ways. It should be noted thatthey can be administered as the compound or as a pharmaceuticallyacceptable salt and can be administered alone or as an active ingredientin combination with pharmaceutically acceptable carriers, diluents,adjuvants, and vehicles. The compounds can be administered intra orsubcutaneously, or peri or intralymphatically, intranodally orintrasplenically or intramuscularly, intraperitoneally, andintrathorasically. Implants of the compounds also can be useful. Thepatient being treated is a warm-blooded animal, and in particular,mammals including man. The pharmaceutically acceptable carriers,diluents, adjuvants, and vehicles as well as implant carriers generallyrefer to inert, nontoxic solid or liquid fillers, diluents, orencapsulating material not reacting with the active ingredients of theinvention.

The doses can be single doses or multiple doses over a period of severaldays.

When administering the compound of the present invention parenterally,it is generally formulated in a unit dosage injectable form (solution,suspension, emulsion). The pharmaceutical formulations suitable forinjection include sterile aqueous solutions or dispersions and sterilepowders for reconstitution into sterile injectable solutions ordispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol (PEG), and the like),suitable mixtures thereof, and vegetable oils. It is notable that PEGinduces a chemically modified (NCE) cytokine preparation (Pegylation).

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such as cottonseed oil, sesame oil, olive oil, soybean oil,corn oil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, can be also used as solvent systems for compoundcompositions. Additionally, various additives that enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, and bufferscan be added. Prevention of the action of microorganisms can be ensuredby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it isdesirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

Peptides may be polymerized or conjugated to carriers such as ovalbumenor human serum albumen as is well known in the art.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicles, additives, and diluents;or the compounds utilized in the present invention can be administeredparenterally to the patient in the form of slow-release subcutaneousimplants or targeted delivery systems such as monoclonal antibodies,vectored delivery, iontophoretic, polymer matrices, liposomes, andmicrospheres. Examples of delivery systems useful in the presentinvention include: U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;4,439,196; and 4,475,196. Many other such implants, delivery systems,and modules are well known to those skilled in the art.

Generally, the initial dose of NCM may be administered eithersimultaneously with thymosin α1 or by administering one drug followed bythe other, generally, and preferably, on the same day. The NCM isadministered at low doses (200-500 units) of IL-2 equivalence as it isimportant not to use high doses (>1000 units/dose) as effect is lost andtoxicity increases.

More specifically, the foregoing provides a protocol for using NCM as anadjuvant to immunize cancer patients against tumor antigens, eitherautologous or as defined proteins or peptides.

The antigen preparations to be used In Cancer 1) PSMA peptides-obtainedcommercially Prostate 2) MAGE 1 & 3 & MAGE fragments & NY ESO-1 Melanomaobtained from the Ludwig Inst of Immunol H&NSCC 3) Papilloma virus E6 &E7 obtained commercially Cervical SCC

The route of antigen plus NCM+thymosin α1 administration ispreferentially the neck because it is accessible and it contains greaterthan 30% of the body's lymph nodes and systemic immunity can beenvisioned to result.

Low dose cyclophosphamide has been used to augment cellular immunity anddecrease suppression by lymphocytes in mice and patients with cancer(Berd, D, Prog in Clin Biol Res 288:449-458 (1989); Berd, D, et al.,Canc Res 47:3317-3321 (1987)) and it has been employed in effectiveimmunotherapy of cancer patients (Weber, J. Medscape Anthology 3:2(2000); Murphy, G P, et al., The Prostate 38:43-78 (1999); Hadden, J W,et al., Arch Otolaryngol Head Neck Surg 120:395-403 (1994)).

Zinc deficiency is associated with improved cellular immunity andtreatment with zinc is immunorestorative in mice (Hadden, J W, JImmunopharmacol 17:696-701 (1995); Saha, A. et al., Int'l JImmunopharmacol 17:729-734 (1995)).

A cyclooxygenase inhibitor (COXi) such as indomethacin is used. Cancersproduce prostaglandins and induce host macrophage production ofprostaglandins (Hadden, J W, The Immunopharmacology of Head and NeckCancer: An Update, Int'l J Immunopharmacol 11/12:629-644 (1997)). Sinceprostaglandins are known to be immunosuppressive for T cells, inhibitionof PG synthesis with cyclooxygenase inhibitors is appropriate.

Recombinant Protein Purification

Marshak, et al., Strategies for Protein Purification andCharacterization, a Laboratory Course Manual, CSHL Press (1996).

Dose and Frequency of Antigens

1-1000 μg, preferably 10-50 are used. The form of antigen is soluble(partially polymerized or conjugated to carrier, only if necessary).

Schedule: Day 1, Day 12, Day 21

(Pre-Rx) Day 12, Day 21, Day 31

Site of Injection: local injection, i.e., neck injections

Expected responses are tumor reduction and tumor pathological changesreduction, fragmentation, lymphoid infiltration). Humoral immunity toantigen (RAI or ELISA) is expected, as well as cellular immunity toantigen (intracutaneous skin test in vitro lymphocyte proliferation orELISPOT ASSAY).

Oligopeptides such as PSMA, MAGE fragments, E6 and E7 peptides would notnormally be immunogenic without conjugation to carrier or pulsed on todendritic cells. Thus, effective immunization would not be expected tooccur. Even with effective immunization, tumor regression would beconsidered surprising by this method, particularly at a distance as withprostate and cervix. Regression of metastatic disease is always asurprising event with immunotherapy. Degree and frequency of clinicalresponses are factors in the effectiveness and thus the novelty of thisapproach. Diagnostic skin tests are another guide to more effectiveimmunization. Patients can be pretreated with IRX-2 (NCM) and thymosinα1 to induce better responses (increase NCM and PHA skin tests andlymphocyte counts and reversal of lymph node abnormalities).

This creates an adjuvant strategy:

-   -   1) Combining immunorestoration and adjuvancy;    -   2) Making peptides and proteins immunogenic;    -   3) Obtaining the degree of immune response to effect tumor        regression at a distance; and    -   4) Extending to all forms of tumor antigens and haptens        including peptides and/or carbohydrates.

It can extend to areas of applicability as in AIDS virus vaccine in HIVpositive patients; other difficult to manage situations; renaltransplants, aged, etc.

Patients are HLA matched for MHC restricted peptides and skin tested forone or more tumor peptides prior to consideration of the protocol. 100μg of one or more tumor peptides are perilymphatically administered inthe neck with NCM plus thymosin α1 using the NCM protocol as discussedbelow on days 1 and 10 of the NCM series. The combination is repeated onday 21. In addition to tumor response and histology, immune reaction tothe peptides is monitored by repeat skin test or by other means known inthe art.

The following examples demonstrate the utility of the present inventionto provide immune restoration and adjuvant effect of NCM plus thymosinα1. As an introduction to the examples, reference is made to U.S. Pat.No. 5,632,983 ('983 patent), the patent having the same inventor as theinventor herein. The '983 patent presents data resulting from in vivotreatments on thymocyte (FIG. 21) and splenocyte (FIG. 22). The datarepresent in vitro response to stimulation with media control (openbars), rIL-1 (solid bars), rIL-2 (cross-hatched), and NCM (diagonallines). Thymosin α1 and NCM alone increase many of the responses in boththe central lymphoid organs. The combination produced dramatic andhighly significant increases for all four responses. This data was onlypresented in vivo in mice.

Example 1

All steps relating to cell culture are performed under sterileconditions. General methods of cellular immunology not described hereinare performed as described in general references for cellular immunologytechniques such as Mishell and Shiigi (Selected Methods in CellularImmunology (1981)) and as are known in the art.

Preparation of Natural Cytokine Mixture (NCM)

The buffy coat white cells of human blood from multiple HIV-negativehepatitis virus-negative donors is collected. In an alternativeembodiment, animals are the cell source for veterinary uses. The cellsfrom the donors are pooled and layered on ficoll hypaque gradients(Pharmacia) to yield lymphocytes free of neutrophils and erythrocytes.Alternative methods could be used that would result in the same startinglymphocyte population as are known in the art.

The lymphocytes are washed and distributed in X vivo-10 media (WhittakerBioproducts) to surface activated cell culture flasks for selection ofcell subsets (MICROCELLECTOR™ T-25 Cell Culture Flasks), which containimmobilized stimulants, i.e., mitogens like PHA. In one set ofexperiments, X vivo-15 and X vivo-20 media were used as indicated. Theimmobilization process for the stimulants is as described by themanufacturer for immobilizing various substances for panning procedures,i.e., separating cells, in the flasks. Alternatively, the lymphocytesare exposed to stimulants e.g., PHA for 2-4 hours then washed threetimes.

The cells are incubated for 24 to 48 hours in X vivo-10 media with 80μg/ml Ciprofloxacin (Miles Lab) at 37° in a CO2/air incubator.Alternatively, RPMI 1640 media could be used (Webb, et al. (1973)).Generally, the HSA is used at 0.1 to 0.5% (weight by volume). Followingincubation, the supernatants are poured off and collected. Human serumalbumin (HSA) may be added to stabilize further the interleukins ifHSA-free media is used for generations. The supernatants are store at 4°C. to −70°.

Characterization of Supernatants

The pooled supernatants are characterized by measuring the cytokinecontent by bioassay for IL-2 and ELISAs for the remaining interleukins:IL-1, IL-15, CSFs, TNFs, and IFNs. Sterility is tested by culture inthioglycolate broth and endotoxin measured by limulus lysate assay as isknown in the art.

Standardization of Supernatant of Cytokine Content:

Each supernatant is standardized either by concentration or amountadministered so that comparisons can be made.

Removal of Contaminants from Supernatant:

DNA and virus exclusion, if used, employ such techniques asultrafiltration, column chromatography, virus retentive filters, ethanolfractionation, polyethylene glycol/bentonite precipitation, gammairradiation, and/or solvent/detergent treatment as has been used forintravenous gamma globulin and monoclonal antibodies (e.g., IGIV NewsUpdate brochure).

Model

The model of hydrocortisone induced thymic involution in aged mice wasused unless otherwise indicated (Hadden J W, et al., Int'l JImmunopharmacol 17:821-828 (1995)).

Laboratory Animals

Female BALB/c (Life Science, St. Petersburg, Fla.) aged retired breedermice (8-9 months) whose thymuses had begun to involute were employed inin vivo tests. Mice were weight matched and randomly pooled in groups offive. Animals were fed standard laboratory diets with drinking water adlib. All mice, with exception of a control group, were treatedintraperitoneally (i.p.) with hydrocortisone (5 mg/mouse in 0.1 ml 0.9%sodium chloride) for two consecutive days to induce a chemicalthymectomy and reduction of spleen weight.

Hydrocortisone-treated adult mice show acute thymic involution (lessthan 30% of control) and reduction in spleen size (less than 80% ofcontrol) at two days with progressive recovery to ten days.

Experimental Design

Each treatment group had five animals and each experiment was repeatedtwo to five times. Treatment was initiated intraperitoneally (i.p.) onday 3 and continued once per day for a total of five days. Treatmentgroups were injected with one of the following in vivo treatments asindicated in the text:

1. pyrogen free saline (controls);

2. recombinant interleukin-1 (rIL-1; 4 ng);

3. recombinant interleukin-2 (ft-2; 50 units);

4. rIL-1+rIL-2 (4 ng+50 units, respectively);

5. natural cytokine mixture (NCM; 50 units IL-2 equivalence)

On day 8, the mice were weighed, sacrificed by cervical dislocation, andtheir spleens and thymuses removed and weighed. The organs were minced,the residual erythrocytes were lysed using ammonium chloride (Mishelland Shiigi (1981)), and the cells counted.

The proliferative response of the cells to various substances was thendetermined. A sample of cells was prepared for cell culture at 37° C.,5% CO2 in RPMI 1640 medium with 5% fetal bovine serum, penicillin (100U/ml), streptomycin (100 μg/ml) and 2-mercaptoethanol (2×10-5 M). Thecells were plated in 0.2 ml microwell plates in quadruplicate at aconcentration of 1.5×106/ml and incubated for 72 hours with one of thefollowing as indicated in the text:

1. control diluent (complete RPMI 1640 medium);

2. rIL-1 (1 ng/ml);

3. rIL-2 (2 Units/ml);

4. NCM (2 Units/ml of IL-2 equivalence);

5. concanavalin A (ConA; 1.5 μg/ml);

6. phytohemagglutinin (PHA; 0.5 μg/ml)

The culture was terminated to measure DNA synthesis, and thereby cellproliferation, with an 18-hour pulse of tritiated thymidine(3H-Thymidine; New England Nuclear, Boston, Mass.; specific activity 6.7Ci/mM), harvested with a multiple automatic sample harvester andprocessed for liquid scintillation counting. Marker studies were alsoperformed as described by Hadden, et al., (1992). The results wereexpressed as arithmetic mean of cpm from three samples for each animal.In order to simplify the representation of data obtained with differentanimals, the results with the different animals were pooled andcalculated together and in some cases are expressed as ratio to controland others as means+brackets for standard error of the mean (SEM).

Statistical Analysis

Student's T test was used to analyze data as appropriate.

Results

The objective was to find a way to stimulate lymphocytes to produce highlevels of interleukin-2 in the absence of serum and in a way that didnot yield significant quantities of PHA in the supernatant. To do this,the PHA was immobilized on surface activated cell culture flasks forselection of cell subsets (AIS MICROCELLECTOR™ T-25 plates) as describedin the manufacturer's instructions for “panning” cell separation orpulsed into the cells followed by washing (pulse technique).

Media employed in these experiments was X vivo-10 (Whittaker) and isapproved for administration to humans by the U.S. Food and DrugAdministration for interleukin-2 lymphokine activated killer (LAK) cellprotocols. Serum-free media capable of supporting human lymphocyteproliferation like minimal essential media (MEM) or RPMI-1640 (Sigma)could also be used.

Initial experiments indicated that PHA (HA-16, Murex Diagnostics Ltd.,Dartford, UK) could be immobilized by the technique described by themanufacturer and that under appropriate optimal conditions of cellnumber of 7.5-15×106/ml, time of exposure of 24 hours to 48 hours, andPHA concentration of 25 or 50 μg/ml a high yield of IL-2 in theserum-free supernatant could be obtained. The yield was superior to thepulse technique employing brief exposures to PHA (NI) followed bywashing and subsequent culture with Ciprofloxacin (NIM) in serum-freemedia (Table I). Therefore, this flask procedure is used to generate theNCM mixture.

TABLE I IL content of supernatant/ml PHA brief exposure (NI) 2-20 unitsPHA brief exposure 8-140 units & Ciprofloxacin (NIM) (80 μg/ml) PHAflask immobilization 100-353 units & Ciprofloxacin (80 μg/ml) IL-2content was measured in the supernatant using the CTLL IL-2 dependentcell line by the methods described by Gillis et al. (1978). IL-2 wasquantitated in international units against a known standard containing640 units (Pharmacia AB).

The cell-free supernatants from flasks incubated without cells weretested on human lymphocytes to determine if residual PHA was present insufficient quantities to produce a proliferative response. Any residualPHA greater than 0.01 μg/ml would give such a response. In the absenceof cells, small amounts of PHA were observed in the supernatant at 40 to48 hours; however, when PHA (25 μg/ml) was used for only 24 hours, theselevels were negligible. Twenty-four hours incubation was thus consideredoptimal.

A comparison of X vivo-10, X vivo-15, and X vivo-20 (Whittaker) and MEMin the present invention was undertaken and shown in FIGS. 1-3. Xvivo-10 and X vivo-15 are approved for administration to humans by theU.S. Food and Drug Administration for IL-2 lymphokine activated killer(LAK) cell protocols. Generation of NCM was compared in different mediautilizing continuous versus pulsed exposure to PHA at 1 μg/ml (FIG. 1).The effect of cell concentration was explored with continuous exposureto PHA at 1 μg/ml (FIG. 2) and PHA at 2 μg/ml (FIG. 3). The optimalcombination of these factors was found to be continuous exposure byimmobilization in X vivo-10 at cell concentrations of 2.5 or 5.0×106/mlwith PHA at 2 μg/ml or at 5×106 cells/ml with PHA at 1 μg/ml. Becausethe per cell yield is most efficient at 2.5×106 cells/ml, thatconcentration with PHA at 2 μg/ml is chosen as the optimal.

Preliminary experiments, in tubes rather than flasks, were performed todetermine the parameters for Ciprofloxacin and two other4-aminoquinolone antibiotics (Norfloxacin and Ofloxacin) to enhancecytokine production from human leukocytes following exposure to PHA.Table II shows that 80 μl/ml of each of these 4-aminoquinoloneantibiotics enhanced production of IL-1, IL-2, IL-6, IFN-γ, TNF-α, andG-CSF. IL-8 production was maximal. IL-3, IL-4, and IL-7 wereundetectable under these circumstances in all supernatants. Theseresults indicate that under these serum-free conditions, all4-aminoquinolones tested at 80 μg/ml enhanced PHA induced cytokineproduction under serum-free conditions.

TABLE II PHA Alone & PHA & Ciprofloxacin Norfloxacin Ofloxacin PHA & PHAIL-1-β 81 1080 783 810 IL-2 ND 120 32 82 IL-6 1665 >3000 >3000 >3000IL-8 18000 >18000 >18000 >18000 IFN-γ ND 750 210 380 TNF-α 54 1935 15004000 GM-CSF 114 4.5 4.5 72 G-CSF 41 555 800 630 Units for cytokinesother than IL-2 are pg/ml and for IL-2 international unit/ml.

It was also determined that a monoclonal antibody, OKT-3 (Ortho), whichinduces T lymphocytes to proliferate and produce interleukins could beemployed as a stimulant under these conditions. Table III shows thatOKT-3 induced cytokines similar to those induced by PHA plusCiprofloxacin with cells incubated in flasks as set forth in Example 1.IL-3, 4, 5, and 7 were not detected with either set of stimulants. OKT-3produced a small additive effect for several ILs when joined with PHAand Ciprofloxacin (CIPRO).

TABLE III CIPRO OKT-3 + CIPRO + PHA + PHA OKT-3 IL-1-β 1080 1530 1125IL-2 120 340 ND IFN-γ 750 4660 11280 IL-6 >3000 >3000 1980IL-8 >18000 >18000 >18000 TNF-α 1935 2700 2500 GM-CSF 4.5 12 75 G-CSF555 375 ND Units of interleukins other than IL-2 are pg/ml and for IL-2international units/ml. ND not done.

In order to show the superiority of the NCM over rIL-1 in vitro, mousesplenocytes and thymocytes were cultured with MEM and rIL-2 atcomparable levels of IL-2 as determined by bioassay and DNA synthesismeasured by tritiated thymidine incorporation. NCM induces greaterproliferation of splenocytes (FIG. 4) and thymocytes (FIG. 5) than rIL-2based on IL-2 content.

In a series of experiments as set forth in FIGS. 6 and 7, mice withinvoluted thymuses were treated in vivo with rIL-1, rIL-2, combinationsof these factors, NCM or saline (controls). The spleens and thymuseswere removed, the cells tested for cell proliferation responses againstthe interleukins (IL-1, IL-2), NCM and the mitogen ConA. The results areexpressed as ratio to the saline treated control. In vivo treatment withrIL-1, rIL-2, and their combination (rIL-1 and ft-2) had no significanteffect to increase proliferative responses of splenocytes (FIG. 6) or ofthymocytes (FIG. 7) to in vitro stimulation with IL-1, IL-2, NCM orConA. NCM treatment in vivo augmented significantly both splenocytes andthymocytes to all 4 stimuli. These results are consistent with anenhanced sensitivity of these cells to stimulation and/or an increase inthe number of responsive cells.

FIGS. 8 and 9 demonstrate the effect of NCM treatment in vivo onsplenocyte and thymocyte markers. Nonmature T cells are indicated by −−and may represent T lymphocyte precursors particularly in the thymus.NCM increased proportionately this population in spleen and thymus.Immature T cells are indicated by ++ and this population isproportionately decreased in thymus by NCM treatment. Mature T cells areindicated by CD4+ and CD8+. NCM increased the proportions of mature Tcells in thymus and their number in spleen. These results are consistentwith an effect of NCM to increase T cell precursors and to promote theirdevelopment to mature T cells in thymus.

FIGS. 10 and 11 demonstrate the splenocyte and thymocyte responses invitro to media (RPMI), rIL-1 (IL-1), ft-2 (IL-2), or NCM after treatmentin vivo with control media or NCM in the hydrocortisone model. The micewere treated as described hereinabove. These data demonstrate that NCMaugments background splenocyte responses, splenocyte responses to IL-1and IL-2, but not NCM and background thymocyte responses and thymocyteresponses to IL-1, IL-2, and NCM.

FIGS. 12 and 13 demonstrate the splenocyte and thymocyte response invitro to ConA and PHA after treatment in vivo with control media or NCM.The mice were treated as described hereinabove.

The in vitro studies demonstrate the superiority of NCM over ft-2 atequivalent doses in sensitizing splenocytes and thymocytes toproliferation signals. The effects on thymocytes reflect promotion ofdifferentiation as well. The NCM composition, but not rIL-1, ft-2, northeir combination, potently promotes in vivo T lymphocyte function (ILresponses) and development (mitogen responses and cell markers) that istherapeutically relevant in any therapeutic measures requiringstimulation of the immune system or restoring even partial functioningof a damaged or defective immune system. For example, chemotherapeuticagents can damage cells, including T lymphocytes, involved in the immuneresponse. The present invention, by stimulating the T lymphocytefunctioning and development, can restore, either partially or entirely,this feature of the immune system if damaged.

Example 2

There is shown that local perilymphatic injections in the neck havingNCM plus low dose cyclophosphamide, indomethacin, and zinc inducedclinical regressions in a high percentage of patients with squamous cellhead and neck cancer (H&NSCC) (Hadden J W, et al., Arch Otolaryngol HeadNeck Surg 120:395-403 (1994); Meneses A, et al., Arch Pathol Lab Med122:447-454 (1998); Barrera J, et al., Arch Otolaryngol Head Neck Surg126:345-351 (2000)) with evidence of improved, recurrence-free survival.Overall, including minor response (25%-50%) tumor shrinkage andreduction of tumor in pathological specimens, over 90% responded and themajority had greater than 50% tumor reduction.

These responses were speculated to be mediated by immune regressionsince both B and T lymphocytes were observed infiltrating the tumors.The therapy was not associated with significant toxicity.

Several unpublished observations serve to document this speculation andlead to the present invention.

Treatment of lymphocytopenic cancer patients with the combination of NCMhas resulted in marked lymphocyte mobilization; where analyzed, thesepatients showed increases in CD45RA positive T cells (i.e., naïve Tcells (Table I)).

Intratumoral or peritumoral injection of NCM in patients with H&NSCCresulted in either reversing immunotherapy-induced tumor regression orin progression of the tumor. The tumor is thus not the site ofimmunization.

Analysis of regional lymph nodes revealed unpublished data that indicatethat the regional lymph node is the site of immunization to postulatedtumor antigens (see FIGS. 14 to 18).

None of these patients treated with NCM developed metastasis expected in15% clinically and up to 50% pathologically, indicating systemicimmunity rather than merely local immunity had been induced.

Patients were pre-tested with a skin test to 0.1 ml of NCM prior totreatment. More than 90% of those with a positive skin test (70.3 mm at24 hours) had robust clinical and pathological response. Patients withnegative skin tests had weak or no response. This skin testing appearsto select good responders.

Major increases were observed in T lymphocyte counts (CD2) 752→1020 inthese T lymphocytopenic patients (T cell counts 752 versus normal=1600).Importantly, there was a corresponding increase in “naïve” CD45RApositive T cells (532→782). As mentioned previously, these increases aregenerally not thought to occur in adults, particularly with apharmacological therapy like NCM. These cells presumably are recentthymic émigrés and could be considered a major new capacity forresponding to new antigens like tumor antigens. The pre-existing CD45RApositive cells were not responding to the tumor antigens and may well beincapable of doing so due to the tumor-induced immune suppression(anergy).

The literature (Hadden J W, Intl J Immunopharmacol 11/12:629-644 (1997);Hadden J W, Intl J Immunopharmacol 21:79-101 (1999)) indicates that forboth SCC and adenocarcinomas, the two major types of cancer, regionallymph nodes reflect abnormalities related to the tumor, including sinushistiocytosis, lymphoid depletion, and often, the presence oftumor-associated lymphocytes capable of reacting to tumor cells (withIL-2). With metastasis, lymphoid depletion and depressed function occur.An unpublished analysis of uninvolved cervical lymph nodes 10H&NSCC and10 controls showed reduction in average size and an increase in sinushistiocytosis associated with H&NSCC (FIGS. 14-17).

TABLE IV Treatment of Lymphocyte Phase Patients with H&NSCC with NCM -Increases in Naïve T Cells in Blood (#/mm³) NAÏVE T CELL MARKER PAN TCELL MARKER Patient # PRE POST INCREASE PRE POST INCREASE 1 479 778 +299704 1171 +467 2 938 1309 +371 1364 1249 −115 3 98 139 +41 146 178 +32 4341 438 +97 655 590 −65 5 567 652 +97 453 643 +190 6 658 1058 +400 11181714 +569 7 642 1101 +459 822 1601 +779 MEAN 532 782 +250 752 1020 +269

Following treatment with one cycle of the NCM (IRX-2) protocol (Hadden JW, et al., Arch Otolaryngol Head Neck Surg 120:395-403 (1994); MenesesA, et al., Arch Pathol Lab Med 122:447-454 (1998); Barrera J, et al.,Arch Otolaryngol Head Neck Surg 126:345-351 (2000)), the uninvolvedcervical lymph nodes showed the changes indicated in FIGS. 14 to 17.Compared to the regional lymph nodes of patients with H&NSCC not treatedwith NCM, these nodes showed a significant increase in size, T cell areaand density, and decreases in number of germinal centers and sinushistiocytosis and congestion. The lymph nodes of treated patients wereall stimulated and were larger than control nodes with increased T cellarea and density. These nodes were thus not only restored to normal, butalso showed evidence of T cell predominance, a known positive correlatewith survival in H&NSCC (Hadden J W, Intl J Immunopharmacol11/12:629-644 (1997)).

Importantly, when the lymph node changes related to B and T cell areaswere correlated with the changes in their tumors reflecting T and B cellinfiltration, a high degree of correlation was obtained for T cells(p.<0.01) and B cells (<0.01) and overall lymphoid presence (p.<0.001)(FIG. 18). These changes correlate with tumor reduction by pathologicaland clinical criteria. These findings indicate that the tumor reactionsare directly and positively correlated with lymph node changes and thatthe tumor reaction reflects the lymph node changes as the dependentvariable. These findings, taken into conjunction with knowledge abouthow the immune system works in general (Roth, I, et al., Male DImmunology, JB Lippincott Co, Phila, Pa. (1989)), and following tumortransfection with a cytokine gene (Maass G, et al., Proc Natl Acad SciUSA 92:5540-5542 (1995)), indicate that the NCM protocol immunizes thesepatients to yet unidentified tumor antigens at the level of the lymphnodes. No one has previously presented evidence for lymph node changesreflecting immunization with autologous tumor antigens. This constitutesa good starting point for trying to induce immunization with previouslyineffective or poorly effective tumor antigens in an effect to yieldregression of distant metastases.

Example 3

Two patients were treated with lymphoma of the head and neck. Thepatients included were those with head and neck cancer who agreed toparticipate in the protocol. The following scheme was followed:

Before treatment, the patients were skin-tested with NCM 0.1 mlsubcutaneously in the forearm, the region was marked, and 24 hourslater, the test was read. The test was considered positive if theinduction and erythema was equal or larger than 3 mm.

Each cycle of NCM was for 21 days, as follows:

Day 1 Low dose cyclophosphamide (300 mg/m² i.v.) Days 1-21 Indomethacin25 mg p.o. 3 times daily Zinc sulfate 50 mg p.o. once daily Days 3-12NCM 200 units 5 as 1 ml subcutaneously perilymphatic in the neck

Case #1

The patient was a 23-year-old male who presented with a prior history of3 months of the presence of a tumor on the left submaxillary region,with no other symptoms. In the emergency room, he was found to havelymph adenopathy of the left submaxillary triangle of approximately 6.5cm in diameter of a hard consistency, partially fixed at deep levels.The rest of the physical exam was normal. The incisional biopsy showedHodgkin's lymphoma. The lesion was staged ECIIA. A one-cycle treatmentof NCM was given, obtaining a minor response, as the adenopathy reducedin size by 1 cm in diameter. The biopsy report obtained after NCMtreatment showed 60% of the lesion showed normal lymphocyticinfiltration, and the rest of the neoplasia (40%) showed necrosis. Noviable tumor cells were found.

Following this, the patient received radiation treatment in the neck of3600 rads. The patient is currently free of disease.

Case #2

The patient is an 82-year-old male who presented with a two-monthhistory of a painful mid-neck tumor mass as well as a 10-kg loss ofweight. On physical exam, the patient presented with tumor on the rightpalatine tonsil, which was enlarged to approximately 4×3 cm, with anulcer in the center of the tonsil. On the neck, a right submaxillarylymph node measured approximately 2×2 cm and a lymph node mass at levelII and III of approximately 5×5 cm. The rest of the exam was normal. Theincisional biopsy of the tonsil and one of the neck's lymph nodesdemonstrated defined non-Hodgkin's lymphoma mixed, of intermediategrade.

The patient was subjected to two cycles of NCM at the end of which a 1cm reduction in the diameter of the tonsil and neck adenopathy wasobserved. The pathological report post-NCM treatment showed live tumor20%, fragmented and necrotic 30%, and normal lymphocyte infiltration50%.

The patient was given chemotherapy (CHOP) for six cycles and laterexternal radiotherapy (RT) at a total dose of 4600 rads. He recurred ateight months post RT with adenomegaly at the occipital level. Thepatient died three months later with evidence of neck disease.

Example 4

Ten patients with untreated early stage cervical cancer, clinicallystaged IB1, IB2, and IIA were treated with local, perilymphaticinjections NCM as IRX-2 (10 daily injections) followed by radicalhysterectomy at day 21. One day before starting IRX-2, patients receiveda single i.v. dose of cyclophosphamide at 300 mg/m2. Oral indomethacinor ibuprofen and zinc sulfate were administered from days 1 to 21. Theclinical and pathological response, toxicity and disease-free survivalwere evaluated.

All patients completed NCM treatment and were evaluated for response andtoxicity. Clinical response was seen in 50% of patients (3 partialresponse (PR), 2 minor response (MR) (>25%<50% reduction)). Sevenpatients underwent surgery. Pathologically, tumor reduction associatedwith tumor fragmentation was found in five cases. There was a ratherheterogeneous pattern of cell types infiltrating the tumor that includedlymphocytes, plasma cells, neutrophils, macrophages and eosinophils.Treatment was well-tolerated except for severe pain and minor bleedingduring injection and gastric intolerance to indomethacin. After 24months of follow-up, nine patients are disease-free.

This previously unpublished study shows that peritumoral NCM inducesimmune-mediated tumor response in early stage untreated cervicalcarcinoma.

Example 5

Two patients with liver metastasis from primary hepatocellular carcinomawere treated with intrasplenic NCM (1 or injections). The protocol wasotherwise as previously described for the H&NSCC, cervical, or lymphomacases. One patient with advanced hepatocellular carcinoma had a partialresponse confirmed by tomography. No histology is available. The otherhad a partial response confirmed by surgery. Histological exam showedtumor reduction, fragmentation, and lymphoid infiltration.

Example 6

Four patients with squamous cell carcinoma of the penis (human Papillomavirus associated) were treated with the NCM protocol as described above.All four had partial responses clinically and the surgical specimenshowed tumor reduction and fragmentation and lymphoid infiltrationcharacteristic of the H&NSCC cancer patients.

Example 7

Mice were immunized with PMSA peptides conjugated to ovalbumen 100 μg at3 sites (days 1, 14, and 21) with alum (1:1 Vol) as adjuvant (5@) or NCM(20 units IL-2 equivalence) (5@). Animals were skin tested at day 28with ovalbumen (100 μg) (2@) or peptides (100 μg) (3@). Two animalstreated with ovalbumen plus NCM without peptides responded to ovalbumenwith positive skin tests. Two animals treated with ovalbumen plus alumdid not respond. Two of three animals treated with ovalbumen pluspeptides and NCM responded. None of the animals treated with ovalbumenplus peptides and alum responded. Thus, NCM was a superior adjuvant toalum for both tumor peptides and ovalbumen as antigens.

Example 8 Phase I/II Study NCM an NCM+Thymosin α1 in LymphocytopenicPatients

Following radiotherapy patients show marked decline of total lymphocytecounts, CD3 and T lymphocytes including both CD4 and CD8 subsets: (CD4drops more than CD8 so that CD4/CD8 ratio drops from 2 to close to 1).During 18 months follow-up these levels did not recover (Wolf, et al.(1985)). Following NCM treatment of T lymphocytopenic patients prior tosurgery, the lymphocyte counts increased significantly (Verastegui, etal. (1999)).

A series of post radiotherapy T lymphogenic patients were treated withIRX-2 (NCM) (7 patients) or IRX-2 (NCM) and Thymosin α1 (7 patients). Atonset, both groups had mean lymphocyte counts of 800. Patients weretreated daily for 10 days with perilymphatic injections in the neck oraxilla (to avoid irradiated area) with 1 ml IRX-2 (approximately 150units IL-2 by ELISA, 640 by bioassay) or IRX-2 plus thymosin α1, 1.6mg/l ml. Lymphocyte counts and various mononuclear cell subsets(CD2,3,4,8,16,19,25 CD45 RO RA and 56) were analyzed by FACS at day 0and approximately at day 12. The patients treated with IRX-2 showed nochange in mean lymphocyte counts at day 12 (800→700) and no change in:

T cells and T cell subsets counts (not shown) B cell counts (CD19 - notshown) macrophages (CD16 - not shown) non-T, non-B lymphocyte counts:(259--->265) CD45 RA counts: (279--->290)

Overall the seven patients treated with NCM+thymosin α1 showed increasesin:

total lymphocyte counts 800--->914 p = NS non-T, non-B cells 261--->451p < .05 CD45 RA 221--->443 p < .05

Of the seven patients treated with NCM+thymosin α1, four showed markedincreases in mean lymphocyte counts, non-T, non-B lymphocytes counts andCD45RA and CD45RO counts without significant changes in T cells, B cellsor macrophages (FIGS. 19 and 20). The non-T, non-B cells were CD56negative (not NK cells) and correlated well with CD45RA positive.

Four patients showed increases in TLC following treatment and thesepatients were studied for CD2 and were for a prolonged period. They givea clearer picture of maturation process.

At the onset, almost one-third of peripheral blood lymphocytes arenon-T, non-B. With treatment, these data show a progressive increaseearly (10 to 12) days of 450 lymphocytes comprising three populations ofT cells: approximately ½ of these are CD2+CD3CD45RA+CD4+ or CD8+, i.e.,naïve mature T cells; approximately ¼ of the remaining are non-T andnon-B, but CD45RA+ and ¾ of the remaining CD2++CD3−. These latter areimmature T cells. Between two weeks and three-plus months, the TLCincreases persist, CD3 and αBTcr (one patient only) progressivelyincrease as do both CD4 and CD8 signifying a progressive maturation tofully mature T cells. Correspondingly, CD45RO+ cells increase indicatingthat new memory cells have been produced, meaning they have seen antigenand have been immunized.

CD2+CD3CD45RA+CD4+ or CD8+ lymphocytes over a 6-week period

Three patients showed no net gain in total lymphocyte counts (TLC)comparing early treatments with late results (1-3 months) TLC 967→933,however, several observations were of note:

All three showed increases in CD45RA +173 decrease in Non-T Non-B −159increases in CD3 +101 increase in B  +75 No increase in CD4 and CD8 — αBTcr increase (1 patient) +155

These changes signify an internal shift from almost half null cells(meaning non-T, non-B markers) towards early T cells CD3+, CD45RA+, αBTcrR+, CD4−CD8−. The shift involves approximately 170 cells/mm2 orapproximately 20% of the cells; if B cells are included, it involves 25%of the cells. This major internal shift has never been seen before inassociation with immunotherapy. It signifies a major abnormalcirculating population of immature cells (committed at least, in part,to the T cell lineage, perhaps to both, 170-T:75-B) present in thesecancer patients. The treatment of NCM+thymosin α1 induced a major shifttowards more mature T cells, yet they lack CD4 and CD8 characteristic ofmature T cells. The presence of null cells has been noted in cancer andother immunodeficiencies, yet the induction of immature CD4−CD8− T cellsin the circulation has not been observed before.

These data document the induction and maturation of new T cells as aresult of treatment with NCM and thymosin α1. Intravascular transitionalT cells progressed to maturity, i.e.:

CD2+CD45RA+CD3→CD2+CD45RA+CD3+TCR+CD4−CD8→

CD2,3+CD4+ and CD2,3+CD8+subsets.

Where these cells are maturing is a matter of speculation. Normallythese events are thought to occur in the thymus. In general, thesepatients are thought to have involuted thymuses incapable of making newT cells. The composition and method of the present invention apparentlyinduces an increase and mobilization of bone marrow T cell precursors(perhaps to a lesser degree, early B cells) that are either traffickingin and out of the thymus or are differentiating extrathymically. Theprogressive appearance of memory cells is important in indicating thatthese new CD45RA+ naïve T cells are transitioning to CD45RO memory cellsas a response to antigen exposure.

According to the definition of adjuvant to be used in a treatment ofinfectious pathogens or tumors, these features are requisite:

the presence and the generation of “naïve” cells capable of reacting toantigen if T lymphocytopenia is present;

the presence of endogenous or exogenously administered peptides capableof being presented to T cells by mature dendritic cells; and

the action of adjuvant plus antigen in an environment capable ofgenerating immunity, such as the regional lymph node, to yield a robustimmunity particularly of the TH1 type. This cellular immunity or T cellimmunity is considered central in the resistance to most pathogens andtumors.

NCM is capable of, in the strategy with low dose cyclophosphamide and anNSAID such as indomethacin, creating lymph node changes (includingdendritic cell maturation) leading to an immunization to cancer andimmune rejection characterized by tumor reduction and fragmentation anda heavy lymphoid infiltration. The combination with NCM+thymosin α1 isexpected to be even more active.

In the examples below, NCM was employed in combination with thymosin α1plus low dose cyclophosphamide and indomethacin to treat recurrent headand neck squamous cell cancer (H&NSCC). It is notable that recurrentcancers of this type following intense immunosuppression byX-irradiation would be considered by the cancer immunotherapy communitynot amenable to any form of immunotherapy. While NCM was effective topalliate recurrent H&NSCC in several patients, a cure was not considereda possibility.

The next set of examples describe relations to endogenous antigensassociated with tumor and/or chronic/latent infections.

Example 9

Patient was a 68-year-old female smoker who was treated for stage II SCC(T2N0M0) of the tongue with partial glossectomy. Ten months later, thepatient had a 1×1 cm local recurrence at the base of the residualtongue. The patient was treated with the NCM protocol as described abovecontaining 250 units of IL-2 by ELISA, however, in addition, 1.6 mg ofThymosin α1 (Zadaxin/Thymalfin) was administered with each of 10perilymphatic injections of NCM. The patient showed an increase oflymphocyte count from 600 to 900/mm3 with the appearance ofapproximately 300 CD2+CD3CD8+CD45RA+naïve T lymphocytes. No toxicity wasobserved. The tumor underwent a complete clinical regression with nofurther treatment. Histological examination of a locally respectedspecimen showed no tumor cells and a marked lymphoid infiltration. Thisimmune regression exemplifies the antitumor adjuvant potential for theNCM+thymosin α1 mixture in combination with endogenous tumor peptides.

Three additional examples show the action of NCM+thymosin α1 to act asan adjuvant of endogenous pathogen-related antigens.

Example 10

Patient was a 30-year-old female with cervical cancer treated withirradiation. The patient had a long history (>3 years) of condylomaaccuminata (venereal warts) indicative of Papilloma virus infection. Thepersistent lymphocytopenia following x-ray therapy prompted use of NCMin combination with thymosin α1 (250 units IL-2+1.6 mg respectively) for10 daily injections in the axilla (without low dose cyclophosphamide andindomethacin). Three to four weeks following the initiation oftreatment, the condyloma accuminata regressed completely and did notrecur. Lymphocyte counts rose from a low 800 to 1300 mm3 over afour-week period. No other treatment was given.

NCM+thymosin α1 is interpreted to have induced immunity to HPV and thusregression of the venereal warts.

Example 11

Patient is a 56-year-old male with Stage IV H&NSCC gum cancer treatedsuccessfully with the NCM protocol plus surgery (maxillectomy andradiotherapy). Due to persistent lymphocytopenia (lymphocyte count of275/mm3) and oral thrush, the patient was treated with NCM+thymosin α1(250 IL-2 equivalence+1.6 mg respectively) for ten daily perilymphaticinjections in the axilla. His lymphocyte count rose to a high of 1500mm3. Three weeks following the initiation of therapy, he developed amaxillary sialadenitis typical of mumps infection. This resolvedspontaneously without other treatment. The patient also had a reductionin oral thrush (candida infection). In this circumstance, NCM+thymosinα1 is interpreted to eradicate and thus to act as an adjuvant foranother viral antigen, as well as the fungal antigen.

Example 12

A 66-year-old female with H&NSCC cancer of the tongue was treated. As aresult of the radiotherapy, the patient suffered for 1½ years persistentoral thrush (Candidiasis) and lymphocytopenia. The patient was treatedwith 10 daily perilymphatic injections in the neck with the combinationof NCM+thymosin α1 as in the above examples. Following the treatment,the patient's lymphocyte count rose from 800 to 1,200/mm3 and the oralCandidiasis resolved completely without other treatments and did notrecur. The combination of NCM+thymosin α1 is interpreted to be anadjuvant to induce immunity to Candida albicans antigens leading to theresolution of a chronic parasitization by this fungus.

The above four examples exemplify how NCM+thymosin α1 can beadministered with exogenous tumor, viral, or fungal antigens to induceimmunity and resolution of the condition. In the case of the tumorantigen, contrasuppression with low dose cyclophosphamide was necessaryto interfere with tumor-induced immune-suppression to effect animmunization. These preceding examples predict that the combination willsimilarly be an adjuvant with exogenously administered antigens in aclassical adjuvant protocol, i.e., mixed with tumor or pathogen,antigen, or peptides in either the prevention or treatment of cancer orinfection.

The novelty of the foregoing is based on the following three points:

Lymphocyte counts do not rise or only slightly rise followingradiotherapy over 18 months of observation (Wolf, et al., ArchOtolaryngol 111:716-725 (1985));

No one has observed a progressive increase in the circulation of

CD2+CD3−CD45RA+→CD2+CD3−CD45RA+CD4−CD8→

CD2+CD3CD45RA+CD4+ or CD8+ lymphocytes over a 6-week period followingany 2-week treatment period including bone marrow with or without thymustransplantation. This progression of events is thought to occur in thethymus and not to occur in adults as seen here; and

Complete resolution of cancer, two viral infections (one as a benigntumor, venereal warts) and a fungal infection within three weeksfollowing initiation of treatment (as would be expected timewise for animmune response under the circumstances) is completely unexpected.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year, and patents, bynumber. Full citations for the publications are listed below. Thedisclosures of these publications and patents in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology that has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the described invention, theinvention can be practiced otherwise than as specifically described.

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1. A method for unblocking immunization at a regional lymph node by:promoting maturation and activation of dendritic cells in a regionallymph node; and allowing presentation by resulting mature dendriticcells of antigen to T cells to gain immunization of the T cells to theantigen.
 2. A method according to claim 1, wherein said promoting stepis further defined as administering a natural cytokine mixture(NCM)+thymosin α₁ perilymphatically into lymphatics that drain intolymph nodes regional to a lesion to be treated.
 3. A method according toclaim 1, wherein the lesion is a cancerous or non-cancerous persistentlesion.
 4. A method according to claim 3, wherein the non-cancerouspersistent lesion is infectious.
 5. (canceled)
 6. A method according toclaim 1, wherein the antigen is an exogenous antigen.
 7. (canceled)
 8. Amethod of inducing immunization to cancer or persistent lesions by:administering an effective amount of an exogenous antigen and anadjuvant consisting of a NCM+thymosin α₁.
 9. A method according to claim8, wherein said administering step is further defined as administeringan NCM including IL-1, IL-2, IL-6, IL-8, IL-10, IL-12, δIFNIFN-γ, TNF-α,FGM-CSFG-CSF, GM-CSF+thymosin α₁.
 10. A method according to claim 8,wherein said administering step is further defined as injecting theNCM+thymosin α₁ perilymphatically, intranodally, intralymphatically,intrasplenically, subcutaneously, intramuscularly, or intracutaneously.11-16. (canceled)
 17. A method of treating a cancer or other persistentlesion in a severely an immune suppressed patient by administering tothe patient an effective amount of a NCM which acts as an adjuvant toendogenous or exogenously administered antigen from the cancer orpersistent lesion to stimulate an immune response in the patient.
 18. Amethod according to claim 17, wherein said administering step is furtherdefined as injecting an NCM including IL-1, IL-2, IL-6, IL-8, TNFα, andIFN+thymosin α₁.
 19. A method according to claim 18, wherein saidadministering step is further defined as injecting an NCM includingIL-1, IL-2, IL-6, IL-8, TNFα, and IFN-γ, +thymosin α₁.
 20. A methodaccording to claim 17, further including the steps of blockingendogenous suppression of T cells directly or indirectly by theendogenous lesion being treated by codelivering cyclophosphamide and anonsteroidal anti-inflammatory drug (NSAID).
 21. A method according toclaim 17, wherein said blocking and inducing steps are further definedas codelivering cyclophosphamide and a nonsteroidal anti-inflammatorydrug (NSAID).
 22. A method according to claim 21, wherein the NSAIDs areselected from the group including indomethacin, Ibuprofen, rofecoxib,celecoxib and other related treated compounds.
 23. A method of vaccineimmunotherapy including the steps of: inducing production of naïve Tcells; and exposing the naïve T cells to endogenous or exogenousantigens.
 24. A method according to claim 23, wherein said exposing stepis further defined as exposing the naïve T cells to endogenouslyprocessed peptide preparation resident in regional nodes of a patientwho possesses a lesion.
 25. A method according to claim 24, wherein thelesion is cancerous or infectious.
 26. A method according to claim 23,wherein said exposing step is further defined as administering anexogenously produced antigen.
 27. A method according to claim 23,wherein said antigen is otherwise nonimmunogenic peptide.
 28. A methodaccording to claim 23, wherein said exposing step is further defined asimmunizing the naïve T cells with matured peptide presenting dendriticcells at a lymph node distal from a lesion to be treated.
 29. A methodof treating lymphocytopenia by administering an effective amount of NCM.30. A method of inducing immunity by: inducing in vivo maturation ofdendritic cells resulting in effective antigen presentation to naïveuncommitted T cells, leading to clonal expansion of t and B cells,thereby creating immunity in a patient.
 31. A method according to claim29, including the further step of infiltrating into tumors byhematogenous spread leading to tumor destruction.
 32. A method ofinducing immunity by: generating a microenvironment in a regional lympnode allowing effective antigen processing and presentation; anddecreasing cells of the lineage of dendritic cells in the lymph nodesinuses that accumulate in a cancer patient so that antigen becomesimmunogenic for T cells.